Volume 8, Issue 2, Pages 465-471 (August 2001) Distinct Mechanisms Control RNA Polymerase II Recruitment to a Tissue-Specific Locus Control Region and a Downstream Promoter  Kirby D. Johnson, Heather M. Christensen, Bryan Zhao, Emery H. Bresnick  Molecular Cell  Volume 8, Issue 2, Pages 465-471 (August 2001) DOI: 10.1016/S1097-2765(01)00309-4

Figure 1 Occupancy of HS2 by p45/NF-E2 in CB3 Cell Clones Reactivates the Adult β-globin Genes (A) Organization of the murine β-globin locus containing the embryonic/fetal (Ey and βH1) and adult (βmajor and βminor) β-globin genes. HS2 resides 40 and 54 kb, respectively, upstream of the βmajor and βminor promoters. Hyperacetylated zones are denoted by shaded boxes below the locus. A schematic of cis-acting elements within HS2 is shown at the bottom. Note that the arrows linking the LCR and the adult β-globin promoters do not imply looping interactions, as the mechanism of the long-range transcriptional control is unresolved. (B) p45/NF-E2 expression in DMSO-induced CB3, MEL, CB3-1, and CB3-2 cells. The doublet (arrows) represents the p45/NF-E2 isoforms. (C) RT-PCR analysis of β-globin expression in CB3, MEL, CB3-1, and CB3-2 cells. RT, reverse transcriptase. (D) ChIP analysis of p45/NF-E2 binding at HS2. Signals obtained from ChIP were normalized to the signal obtained from input chromatin. The graph depicts data from four independent experiments (mean +/− SEM). PI, preimmune; No Chr, no chromatin. (E) Ethidium bromide-stained gels from a representative experiment Molecular Cell 2001 8, 465-471DOI: (10.1016/S1097-2765(01)00309-4)

Figure 2 p45/NF-E2 Is Dispensable for Histone Acetylation at the LCR but Induces Hyperacetylation Downstream of Its Binding Site (A) ChIP analysis of histone acetylation at HS2 from nine (CB3 and MEL), five (CB3-1 and CB3-2), or two (WEHI) independent experiments (mean +/− SEM). Signals obtained from PCR amplification of immunoprecipitated chromatin were normalized as described in Experimental Procedures. The inset depicts the linearity of input signals for MEL cell chromatin. H3, acetylated histone H3; H4, acetylated histone H4; IgG, immunoglobulin G; PI, preimmune; No Chr, no chromatin. (B) ChIP analysis of histone acetylation at HS3 from four independent experiments (mean +/− SEM). (C) ChIP analysis of histone acetylation at the βmajor promoter from ten (CB3 and MEL), five (CB3-1 and CB3-2), or two (WEHI) independent experiments (mean +/− SEM). (D) ChIP analysis of histone acetylation within the βmajor open reading frame from six (CB3 and MEL) or three (CB3-1 and CB3-2) independent experiments (mean +/− SEM). (E) ChIP analysis of histone acetylation at the βminor promoter from four independent experiments (mean +/− SEM) Molecular Cell 2001 8, 465-471DOI: (10.1016/S1097-2765(01)00309-4)

Figure 3 RNA Polymerase II Associates Selectively with the H3 Hyperacetylated βmajor Promoter ChIP analysis of pol II binding at the βmajor and cad promoters in CB3 and MEL cells. Signals obtained from ChIP were normalized as described in Experimental Procedures. H3, acetylated histone H3; H4, acetylated histone H4; Pol II, RNA polymerase II; IgG, immunoglobulin G; No Chr, no chromatin. (A) Ethidium bromide-stained gels from a representative experiment. (B) Quantitation of ChIP data from two independent experiments for the βmajor promoter. (C) Quantitation of ChIP data from one experiment for the cad promoter Molecular Cell 2001 8, 465-471DOI: (10.1016/S1097-2765(01)00309-4)

Figure 4 Distribution of RNA Polymerase II within the β-globin Locus (A) Organization of the murine Hbbd β-globin locus. (B) ChIP analysis of pol II binding within the β-globin locus in CB3 and MEL cells. Signals obtained from ChIP were normalized as described in Experimental Procedures. Representative ethidium bromide-stained gels are shown for each site examined. The graphs show data from three (IVR2), four (HS1 and Ey promoter), and five (HS3, HS2, and βmajor promoter) independent experiments (mean +/− SEM). Pol II, RNA polymerase II; IgG, immunoglobulin G; No Chr, no chromatin. (C) Western blot analysis of p45/NF-E2 and pol II protein expression in undifferentiated and DMSO-induced MEL cells. (D) ChIP analysis of pol II binding at HS2 and the Ey and βmajor promoters in uninduced and DMSO-induced MEL cells. The graphs show data from one experiment. (E) ChIP analysis of pol II binding at HS2 and the βmajor and cad promoters in WEHI cells. The graph shows data from two independent experiments. (F) Serine 5 phosphorylation of HS2- and βmajor promoter-bound pol II. A representative gel of ChIP analysis with antibodies recognizing pol II and serine 5 phosphorylated pol II (H14) is shown. The ratios of H14/Pol II reactivity are indicated (mean +/− SEM, n = 4) Molecular Cell 2001 8, 465-471DOI: (10.1016/S1097-2765(01)00309-4)